A biosystematic study of seven Afrotropical and two Oriental species of the Imicola species-group was undertaken; this group of Culicoides of the subgenus Avaritia includes C. imicola the most important vector of the viruses of bluetongue (BT) and African horsesickness (AHS) known in the Old World. Five African species are redescribed i.e. C. imicola, C. pseudopallidipennis, C. bolitinos, C. miombo and C. loxodontis. Two new species are described, and the extralimital C. brevitarsis and C. nudipalpis are discussed where relevant. These nine species comprise the Imicola group, one of 10 groups constituting the subgenus worldwide. Due to confusion in the literature, the Imicola group is redefined and distinguished from the Orientalis group (also redefined); 21 species of approximately 70 world species of Avaritia are reassigned to either of the two groups. A key to all nine known species of the lmicola group is given; shortcomings in the taxonomy of the Orientalis group are discussed. The adult morphology of both sexes of the nine Imicola group species was studied; this revealed deficiencies in the descriptive format currently used in taxonomic studies of world Culicoides. Accordingly, greater detail has been introduced into descriptions and includes the use of new character states. Methods for mounting Culicoides on glass slides are also improved to ensure more accurate observation and measurement of diagnostic taxonomic features; furthermore, the descriptions are based on long series of each sex. Illustrations were made from specimens mounted symmetrically, and no feature omitted from any bodypart illustrated. Certain aspects of the life-cycle of most species were investigated but especially those of C. bolitinos, C. loxodontis and C. kwagga. The immatures of these develop exclusively in the dung of the elephant, the buffalo, the blue wildebeest, both species of rhinoceros and Burchell's zebra. Two of these species, C. bolitinos and C. kwagga, have broadened their resource range as they can invade and mature in the dung of cattle and horses. This has obvious implications for the transmission of viruses, especially where indigenous herbivores are run with domesticated livestock. In an intensive two-year survey a comparison was made between the Culicoides fauna of a natural area, the Kruger National Park (KNP), and that found in areas changed by Man, namely livestock farms adjoining the KNP. This part of the eastern Transvaal lowveld is a main focus of AHS in South Africa. Light-trapping, rearing from dung, and pootering off live hosts, revealed that some species of the Imicola group are exclusively associated with certain herbivores; these include the elephant and the zebra which are suspected or proven reservoir hosts for AHS. The results thus throw further light on the epidemiology of this disease, and also show that Man plays a decisive role in determining the numbers, and distribution, of particular Imicola group species under certain conditions. In the case of C. imicola, the commonest and most widespread of all species, this range expansion, or establishment of foci, is due to man's maintenance of domesticated livestock in confined species, and especially where these are kept on irrigated pastures. However, the serendipitous discovery of a large imicola-free zone in South Africa indicates that edaphic conditions likely play an even more important role than Man and climate in determining the prevalence and abundance of C. imicola. This area is the sandy dune field west of Port Elizabeth and holds promise as a natural quarantine zone for the import and export of livestock. AFRIKAANS : 'n Biosistematiese ondersoek van sewe Afrotropiese en twee Orientale spesies van die Imicola groep is gedoen; by hierdie groep Culicoides van die subgenus Avaritia word C. imicola wat as die mees belangrikste vektor van bloutong-(BT) en perdesiekte (AHS) virus in die Ou Wereld beskou word, ingesluit. Vyf Afrika spesies, nl. C. imicola, C. pseudopallidipennis, C. bolitinos, C. miombo en C. loxodontis, is herbeskryf. Twee nuwe spesies is beskryf asook die suid-oos Asiese spesies C. brevitarsis en C. nudipalpis word bespreek waar van toepassing. Hierdie nege spesies vorm die Imicola groep, een van die 10 groepe waaruit die subgenus Avaritia wêreldwyd bestaan. As gevolg van verwarring in die literatuur is die Imicola groep hergedefinieer en geskei van die Orientalis groep ( ook hergedefinieer); 21 spesies van ongeveer 70 wereld spesies van Avaritia is heringedeel in die twee groepe. 'n Sleutel vir al nege wereld spesies van die Imicola groep, asook 'n verspreidingskaart vir elke spesie, word gegee. Tekortkominge in die taksonomie van die Orientalis groep word ook bespreek. Die volwasse morfologie van beide geslagte van die nege Imicola groep spesies is bestudeer; dit het gebreke in die formaat wat tans vir die taksonomiese beskrywing van wêreld Culicoides gebruik word aan die lig gebring. Gevolglik is daar meer data in die beskrywings, wat nuwe karakterkenmerke insluit. Die metode van die montering van Culicoides op glasplaatjies is ook verbeter om meer akkurate ondersoek en meting van die diagnostiese kenmerke te verseker; verder, is beskrywings gebaseer op lang reekse van elke geslag. Illustrasies is gemaak van voorbeelde wat simmetries gemonteer is en geen kenmerk is uitgelaat van enige gelllustreerde liggaamsdeel. Aspekte van die lewensiklus van die meeste van die spesies, veral C. bolitinos, C. loxodontis en C. kwagga is ondersoek. Die onvolwassenes van hierdie spesies ontwikkel slegs in die mis van olifante, buffels, wildebeeste, renosters en zebras. Twee van die spesies, C. bolitinos en C. kwagga, het hulle broeimediums van voorkeur vergroot en kan eiers lê en tot volwassenheid ontwikkel in die mis van beeste en perde. Dit het vanselfsprekende gevolge vir virusoordrag tussen inheemse herbivore en vee, veral in gebiede waar die twee groepe saamloop. In 'n intensiewe twee-jaar studie, is 'n vergelyking gemaak tussen die Culicoides fauna soos aangetref in 'n ongerepte deel van Afrika, die Kruger Nasionale Park (KNP), en in gebiede aangrensend aan die KNP wat deur die mens in veeplase omskep is. Die deel van die Oos Transvaalse laeveld is bekend as 'n perdesiekte "hotspot" in Suid-Afrika. Ligvalvangste, uitbroei van Culicoides uit mis en versameling vanaf lewendige gash ere het aangedui dat sekere spesies van die Imicola groep eksklusief met sekere herbivore geassosieeris; hierby ingesluit is die olifant en zebra wat onderskeidelik verdagte en bevestigde gashere van AHS is. Die resultate dra by tot die verklaring van die epidemiologie van hierdie siekte en wys ook dat die mens 'n beslissende rol speel in die vasstelling van die getalle sowel as die verspreiding van spesifieke spesies van die Imicola groep. In die geval van C. imicola, die mees algemeenste en wydverspreidste spesie, is die uitbreiding, of daarstelling van fokuspunte, te wyte aan die mens se instandhouding van vaste bloedbanke op besproeide weiding. Die toevallige ontdekking van 'n groot imicola-vry sone elders in Suid-Afrika dui daarop dat grondtipe moontlik 'n bepalende rol kan speel in die aanwesigheid en volopheid van C. imicola. Hierdie "skoon" area is die sandduine-veld wes van Port Elizabeth en lyk belowend as 'n natuurlike kwarantyn gebied vir die invoer en uitvoer van lewende hawe. / Dissertation (MSc)--University of Pretoria, 2013. / Animal and Wildlife Sciences / unrestricted

The viral determinants of African horsesickness virus (AHSV) cytopathology are not well understood. Several AHSV proteins may play a role, including non-structural protein NS3, a cytotoxic membrane protein that localises to sites of virus release and plasma membrane disorganisation in infected cells. AHSV NS3 is highly variable and clusters into three phylogenetic groups, termed α, β and γ. In chapter 2 we examined the role of NS3 in determining the phenotypic characteristics observed during AHSV infection of cells. Three AHSV strains, AHSV-2 (γ NS3), AHSV-3 (β NS3) and AHSV-4 (α NS3), were shown to have quantitatively different phenotypes in Vero cells. To investigate the contribution of NS3 to these differences, reassortants were generated between these strains in which the S10 genome segment encoding NS3 was exchanged, alone or in combination with other segments. Exchange of NS3 resulted in changes in virus release and membrane permeability, indicating an important role for NS3 in these viral properties. The cytopathic effect and decreased viability of infected cells was not associated with NS3 alone and it is likely that a number of viral and host factors contribute to these complex phenotypes. In chapter 3 the cytolytic effect of the NS3 proteins of the orbiviruses AHSV, bluetongue virus (BTV) and equine encephalosis virus (EEV) were compared. Inducible expression in Escherichia coli (E. coli) showed differences in cytotoxicity, with EEV NS3 having a greater lytic effect than than AHSV and BTV NS3. Cytotoxicity was linked to increased membrane permeability of the cells as confirmed by an increased uptake of membrane impermeant compounds. When expressed in insect cells however all three NS3 proteins caused a marked but equivalent decrease in cell viability. Although the orbivirus NS3 proteins have similar predicted secondary structures, differences could lie in structural stability and association with membranes of specific cell types, which impacts on cytotoxicity. To determine the regions within AHSV NS3 that mediate cytotoxicity, a series of truncated mutants of NS3 where constructed and expressed in E. coli. The combined presence of both hydrophobic domains of AHSV NS3 was found to be critical for membrane permeabilisation and cytotoxicity. In chapter 4 the AHSV-2, AHSV-3 and AHSV-4 NS3 proteins (from the γ, β and α NS3 clades)were compared to examine the impact of sequence variation in NS3 on structure and function. The proteins were expressed in the baculovirus expression system as both wild-type proteins and C-terminal eGFP (enhanced green fluorescent protein) fusions. Exogenous addition of the baculovirus expressed proteins to Vero cells resulted in different permeabilisation levels that could be linked to that induced by the AHSV strains. Cell viability and membrane association assays in insect cells showed that all three proteins were equivalently cytotoxic and membrane associated. The subcellular localisation of the eGFP-NS3 fusion proteins was examined by confocal fluorescen imaging of live cells. NS3 localised to the plasma membrane, and as distinct punctuate foci in the perinuclear region. This suggests localisation to the internal membrane systems of cells and has important implications for the function of this membrane permeabilising protein. / Thesis (PhD)--University of Pretoria, 2011. / Genetics / unrestricted

Non-structural protein, NS1 of African horse sickness virus is a hydrophobic protein of 63 kDa that spontaneously assembles into highly distinct tubular structures when expressed in mammalian or insect cells. The spontaneous assembly of these proteins into a predictable multimeric structure, high levels of expression and ease of purification make this protein an ideal candidate for the immune display of foreign peptides. The potential of such a display system has been investigated for BTV NS1 that is able to successfully elicit both a humoral and a cellular immune response against inserted peptides. The aims of this study were to investigate both the stability of the AHSV NS1 particulate structure after insertion of peptides as well as the antigenicity and immunogenicity of the peptides presented in this system. Two overlapping regions consisting of 40 and 150 amino acids, and which correspond to a neutralising region identified within the AHSV major neutralising protein VP2, were inserted into an internal site in NS1. This site offered the best surface display of inserted peptides on the tubular structures. An enhanced green fluorescent protein, 240 amino acids long, was also inserted into the NS1 protein. Sucrose gradient analysis of the recombinant proteins indicated that the majority of the baculovirus expressed chimeric proteins formed particulate structures with a sedimentation value similar to that of the native NS1 protein. This was confirmed by transmission electron microscopic analysis, which clearly showed that all the chimeric proteins assembled into tubular structures similar to those observed for AHSV NS1 proteins. Furthermore, fluorescence analysis of sucrose gradients of NS1/eGFP also showed high levels of fluorescence that corresponded directly to particle formation. Not only do the inserts remain functional but are also presented successfully on the surface of the intact NS1 tubule structure. The potential of the NS1 vector to efficiently present peptides to the immune system was subsequently investigated. The serums generated against these chimeric proteins in guinea pigs were tested against chimeric constructs, the baculovirus expressed inserts (for eGFP) and the inserts presented on other presentation vectors. Western blot analysis showed that most of the serums generated against the chimeric proteins contained antibodies not only against the chimeric proteins but antibodies that reacted specifically with the inserted peptides on their own or on another presentation system. Preliminary immune studies seem to indicate that the humoral immune response elicited by the chimeric NS1 proteins is predominantly against the inserts. The inserts are successfully presented to the immune system on the surface of the NS1 vector and are able to elicit the production of antibodies with the potential to provide a protective immune response. / Dissertation (MSc (Genetics))--University of Pretoria, 2010. / Genetics / unrestricted

VP6 is one of the minor structural core proteins of African horsesickness virus. The minor core proteins VP1, VP4 and VP6 are presumed to constitute the dsRNA dependent RNA polymerase transcription complex of the virus. In the Orbivirus prototype bluetongue virus (BTV), VP6 has a helicase activity. The aim of this investigation was to characterize the primary structure and nucleic acid binding function of the inner core protein VP6 of African horsesickness virus (AHSV). To characterize the primary structure of AHSV VP6, VP6 genes of serotypes 3 and 6 were cloned and sequenced. Both genes encode a 369 amino acid polypeptide. A comparison to the VP6 proteins of other Orbiviruses indicated that in all cases the proteins are rich in basic residues and in glycine. The proteins are highly conserved within serogroups but the conservation between serogroups is low. VP6 of AHSV-3 and AHSV-6 have 93.5% identity and 96% similarity in amino acid residues. AHSV-6 VP6 has 27% identical and 46% similar amino acid residues to BTV-10 VP6. Phylogenetic analysis of four orbivirus VP6 genes indicated that AHSV and BTV are most closely related to each other. Motifs characteristic of known helicases were identified by sequence analysis. Glycine rich protein motifs and a N-glycosylation signal were present. No nucleic acid binding motifs identified in other proteins were found in AHSV VP6. To characterize the VP6 protein of AHSV VP6, the genes were expressed using both a baculovirus and a bacterial expression system. Proteins were found to be soluble and the VP6 expressed in insect cells was found to be N-glycosylated. The nucleic acid binding function of AHSV VP6 was investigated. Bacterially expressed VP6 was demonstrated to bind nucleic acids by electrophoretic mobility shift assays. Baculovirus expressed VP6 bound double and single-stranded RNA and DNA in nucleic acid overlay protein blot assays. Competition assays indicated that VP6 may have a preference for binding to RNA rather than DNA. Glycosylation was found to play no direct role in nucleic acid binding but the binding is strongly dependent on the NaCl concentration. A series of truncated VP6 peptides were produced to investigate the importance of localized regions in nucleic acid binding. Two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. Binding appeared to be influenced by charge as reflected by the isoelectric points (pI) of the peptides and experiments indicating the effect of pH on the binding activity. However, only peptides containing amino acid residues 190 to 289 showed binding activity. This region corresponded to the region on BTV VP6 that contains two binding domains. It is proposed that the dsRNA binding domain in AHSV VP6 is a sequence of positively charged amino acids constituting a domain that determines the nucleic acid binding characteristics of the peptide. The mechanism of binding of baculovirus expressed VP6 in a nucleic acid overlay protein blot is proposed to be charge related. / Thesis (PhD (Genetics))--University of Pretoria, 2007. / Genetics / unrestricted

Culicoides (Diptera: Ceratopogonidae) are small (<3mm) blood feeding flies. These flies are biological vectors of viruses, protozoa and filarial nematodes affecting birds, humans, and other animals. Among the viruses transmitted those causing bluetongue (BT), African horse sickness (AHS) and epizootic haemorrhagic disease (EHD) are of major veterinary significance. Culicoides (Avaritia) imicola Kieffer, a proven vector of both AHS and BT viruses, is the most abundant and wide spread livestock-associated Culicoides species in South Africa. Field isolations of virus and oral susceptibility studies, however, indicated that a second Avaritia species, C. bolitinos Meiswinkel may be a potential vector of both BT virus (BTV) and AHS virus (AHSV). Differences in oral susceptibility, which are under genetic control, of populations from different geographical areas to viruses may be an indication of genetic differences between these populations, which may be the result of limited contact between these populations. A good knowledge of the distribution, spread and genetic structure of the insect vector is essential in understanding AHS or BT disease epidemiology. In the present study, an effort was made to gather field specimens of both C. imicola and C. bolitinos from different areas within their natural distribution in South Africa. The aim was to partially sequence two mitochondrial genes from these specimens and to analyse the sequence data making use of phylogenetic trees to clarify the genetic relationships between individuals or groups collected from geographically distinct sites. The two species were collected from four geographically separated areas in South Africa viz. Gauteng Province, Eastern Cape Province, Western Cape Province as well as the Free State Province. DNA was extracted from a total of 120 individual midges of the two Culicoides species using DNA extraction kits. Extracted DNA was analysed using PCR, sequencing as well as phylogenetic methods. A total of 117 mitochondrial DNA COI and 104 mitochondrial 16S ribosomal RNA Culidoides</i. sequences were analysed. DNA sequence polymorphism and phylogenetic relationships of various groups of C. imicola and C. bolitinos midges were determined. The results of the phylogenetic analysis of Culicoides populations using mitochondrial COI gene fragment showed that, at least one subpopulation of C. imicola and two distinct genotypes of C. bolitinos species do exist in South Africa, and further analysis is necessary. This study showed that COI has the potential to separate Culicoides midges based on their geography / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted